Methods for making core/shell nanocrystals that are fluorescent and have hydrophobic surfaces are well known. The hydrophobic surfaces of these nanocrystals typically result from a coating of hydrophobic passivating ligands such as trioctylphosphine (TOP), trioctylphosphine oxide (TOPO), oleic acid, octylphosphonic acid (OPA) or tetradecylphosphonic acid (TDPA) on the surface of the particle. These passivating ligands serve many roles, including, but not limited to: protecting the surface of the nanocrystal by keeping reactive molecules (and even the solvent) away from the nanocrystal surface, protecting the nanocrystal from reactions that could occur when it is in a photoactivated state, preventing the coalescence of multiple nanoparticles, preventing “dangling bonds” and other similar surface defects that could serve as trap sites for an excited electron or hole and thereby promote non-radiative recombination (i.e., reduce the quantum yield), etc. These ligands also provide a layer of alkyl groups that form the solvent-exposed surface surrounding the nanocrystal, and thus render the nanocrystal effectively hydrophobic, regardless of the inherent properties of the nanocrystal surface itself. These intimately associated ligands and the nanocrystal they are associated with form a nanoparticle.
For many applications, it may be desirable to use the layer of ligands on the surface of the nanocrystal to manipulate the properties (e.g., impart hydrophilicity, etc.) of the nanoparticle they collectively form, or to attach other molecules to the nanoparticle. There are conventional methods for doing that are known in the art. For example, one way this has been done in the past involves adding another layer of material over the layer of passivating ligands present on the conventional nanocrystal surface. Adams, et al., U.S. Pat. No. 6,649,138. The second layer is often an amphiphilic polymer (AMP) that is bound to the layer of passivating ligands only by a hydrophobic-hydrophobic interaction between the alkyl groups of the passivating ligands and similar alkyl groups on the AMP polymer. See Adams, et al. However, this effectively makes the resulting nanoparticle larger, increasing the overall size of a nanocrystal by up to 50% or more. For example, a small nanocrystal with a coating of cysteine can be about 6 nm in diameter, yet the same nanocrystal coated with TOPO plus the AMP polymers from Adams, et al. may be up to 15 nm in diameter. See Liu, et al., J. Amer. Chem. Soc., vol. 129, 14530-31 (2007).
The bilayer structure of these AMP-containing nanoparticles can also make the nanoparticle susceptible to degradation that can occur if anything interferes with keeping the layer of passivating ligands on the surface of the nanocrystal, or with the interaction between these passivating ligands and the outer layer of AMP added over them.
For some applications, there are certain advantages to making a nanoparticle as small as possible, especially for certain biological applications. Smaller particles can diffuse more rapidly, have less effect on a molecule they are attached to, and may have less tendency to accumulate in specific tissues in vivo, where larger particles seem to get trapped by ‘filtration’ effects. See, e.g., Ballou, et al., Bioconjugate Chem., vol. 15, 79-86 (2004). Thus better methods for making nanocrystals into water-soluble nanoparticles are needed, preferably methods that keep these nanoparticles as small as possible while making them highly stable and maintaining their essential fluorescence characteristics. This disclosure provides methods for achieving such objectives, and thus provides compositions and methods that produce improved nanoparticles, especially small, stable, water-soluble ones.